Antiviral Effects of Heparan Sulfate Analogue‐Modified Two‐Dimensional MXene Nanocomposites on PRRSV and SARS‐CoV‐2

Due to the worldwide impact of viruses such as SARS‐CoV‐2, researchers have paid extensive attention to antiviral reagents against viruses. Despite extensive research on two‐dimensional (2D) transition metal carbides (MXenes) in the field of biomaterials, their antiviral effects have received little attention. In this work, heparan sulfate analogue (sodium 3‐mercapto‐1‐propanesulfonate, MPS) modified 2D MXene nanocomposites (Ti3C2‐Au‐MPS) for prevention of viral infection are prepared and investigated using severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pseudovirus and porcine reproductive and respiratory syndrome virus (PRRSV) as two model viruses. Ti3C2‐Au‐MPS nanocomposites are shown to possess antiviral properties in the different stages of PRRSV proliferation, such as direct interaction with PRRS virions and inhibiting their adsorption and penetration in the host cell. Additionally, Ti3C2‐Au‐MPS nanocomposites can strongly inhibit the infection of SARS‐CoV‐2 pseudovirus as shown by the contents of its reporter gene GFP and luciferase. These results demonstrate the potential broad‐spectrum antiviral property of Ti3C2‐Au‐MPS nanocomposites against viruses with the receptor of heparin sulfate. This work sheds light on the specific antiviral effects of MXene‐based nanocomposites against viruses and may facilitate further exploration of their antiviral applications.

Ultraviolet visible absorption spectra (UV-Vis) were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). The dynamic light scattering (DLS) and Zeta potential analyses were performed using the Malvern Zeta sizer instrument (Malvern ZEN 3690). The elemental and structural analysis of the samples was performed using an ESCALAB Xi † photoelectron spectrum (XPS) instrument (Thermo Fisher Scientific, USA). The Fourier transform infrared spectra (FT-IR) were recorded by a Nicolet Avatar-330 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA). The confocal fluorescence images were obtained by an Olympus IX 70 inverted microscope (Olympus, Japan).

Cells and viruses
MARC-145 cells, HEK-293T cells, and HEK-293T-ACE2 cells (ACE2-expressing 293T cells) were cultured in DMEM/HIGH GLUCOSE (HyClone) containing 10% fetal bovine serum (FBS, PAN) at 37°C with 5% CO2 in a humidified incubator. PRRSV strain WUH3 (GenBank accession no. HM853673), a highly pathogenic type 2 (North American) PRRSV, was previously isolated from the brains of pigs suffering from a high fever syndrome in China at the end of 2006 [1] . PRRSV was amplified, and the titer was determined in MARC-145 cells by plaque reduction assay.

Plaque reduction assay
To determine viral titers, a plaque reduction assay in MARC-145 cells was performed. Briefly, MARC-145 cells were seeded in 6-well plates and cultured until ~100% confluence and then infected with 800 μL of 10-fold serial dilution of virus. After incubation for 1 h, the nonadherent virions were removed, followed by three washes with PBS, and overlaying the cells with 48% low melting agarose (Biofrox, w/v: 1.8%) in DMEM without phenol red (Gibco) supplemented with 3% FBS and 1% kanamycin-streptomycin. Then the 6-well plate was placed at 4 ℃ for 15 min to coagulate the agarose, followed by incubation at 37 ℃ for 2-4 days, and staining the cells with neutral red (3-Amino-7dimethylamino-2-methylphenazine hydrochloride, Sigma) solution (0.50 mgmL -1 ) for 1 h at 37 ℃ .
Finally, cells were washed and viral titers were determined by counting the number of plaques and presented in plaque forming units (PFU/mL).

Western blot assay
After seeding in 6-well plates and culture until ~80% confluence, the MARC-145 cells were treated as described, followed by washing the cells three times with precooled PBS, suspension in lysis buffer (Beyotime, P0013) with protease inhibitors for indicated periods of time, incubation on ice for 10 min and then centrifugation at 12,000 rpm and 4 ℃ for 10 min. After cell lysis, equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% SDS), and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.22 μm, Millipore, Billerica, MA). After blocking for 4 h with 10% (w/v) nonfat dry milk in Tris-buffer containing Tween 20 (TBST), the membranes were incubated with various antibodies, including primary antibodies against PRRSV N protein and PRRSV nsp2 protein for 4 h at room temperature, with β-actin as the internal control (1:1000, A1978, Sigma-Aldrich). After three washes with TBST, the membranes were incubated for 1 h at room temperature with corresponding horseradish-peroxidase (HRP)-conjugated secondary antibodies (Beyotime) against mouse primary antibodies. After another four washes with TBST, the membranes were detected by the ChemiDocTM imager (Bio-Rad) and analyzed using the Image LabTM software (Bio-Rad).

Indirect immunofluorescence assay
After seeding on circular glass coverslips in 24-well plates, the MARC-145 cells were treated with GFP-PRRSV at a MOI of 1.0. At the indicated time points post infection, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with precooled methanol for 10 min. After blocking with 5% BSA for 1 h, cells were incubated with 4, 6-diamidino-2-phenylindole (DAPI; Beyotime) (5 μgmL -1 ) in PBS for 15 min. Finally, the fluorescent images were acquired with an Olympus FV10 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR)
After seeding in 24-well plates and culture until ~80% confluence, the MARC-145 cells were treated as described, followed by extracting the RNA from the samples at indicated time points by using RNA-Solv® Reagent (Omega Bio-tek), and reverse transcription of 1 μg RNA to cDNA with the Transcriptor First Strand cDNA synthesis kit (Roche, Mannheim, Germany) using random primers as instructed by the respective manufacturers. Subsequently, the resulting cDNA was used as the template in a SYBR green qPCR assay (Applied Biosystems), using (primers specific to PRRSV ORF7 gene as well as the PRRSV ORF7 gene) forward and reverse primers of q5′ORF7-F: GCATTTGTATTGTCAGGAGC-3′ and q5′ORF7-R: AGCAGTGCAACTCCGGAAG-3′, respectively [2] . Each sample was assayed three times. The abundance of individual mRNA transcripts in each sample was assayed three times and normalized to that of GAPDH mRNA (internal control). The forward and reverse primers for the MARC-145 cell reference gene were q5′GAPDH-F: TCATGACCACAGTCCATGCC and q5′GAPDH-R: GGATGACCTTGCCCACAGCC.